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Lymphatic filariasis and onchocerciasis are major neglected tropical diseases affecting over 90 million people worldwide with painful and profoundly disfiguring pathologies (such as lymphoedema or blindness). Type 2 inflammation is a hallmark of filarial nematode tissue infection and is implicated both in eosinophil dependent immunity and lymphatic or ocular immunopathologies. Type-2 innate lymphoid cells (ILC2) are known to play an important role in the initiation of type 2 inflammation in helminth infection. We therefore tracked comparative IL-12Rβ2+ILC1, ST2+ILC2 and NKp46+natural killer (NK) innate lymphoid cell population expansions duringBrugia malayiexperimental peritoneal filarial infections using either immunocompetent or immunodeficient mice. In immunocompetent BALB/c animals, NKp46+NK cells rapidly expanded representing over 90% of the ILC population in the first week of infection, whereas, surprisingly, ST2+ILC2 failed to expand. NKp46+NK cell expansions were confirmed in RAG2 deficient mice lacking adaptive immunity. Ablation of the NKp46+NK cell compartment in RAG2 common gamma chain (gc) mice led to increased susceptibility to chronic adultB. malayiinfection. This data was recapitulated using anOnchocerca ochengimale worm peritoneal implant model. When NKp46+NK cells were depleted in RAG2 deficient mice using anti-NKp46 or asialo GM1 antibody injections over the first five weeks ofB. malayiinfection, susceptibility to adultB. malayiinfection was significantly increased by 2-3 fold with concomitant impairment in eosinophil or neutrophil recruitments. Finally, we demonstrate that in RAG2 deficient mice, drug clearance of a primary adultB. malayiinfection followed by challenge infection leads to resistance against early larvalB. malayiestablishment. This innate resistance is associated with bolstered NK and eosinophils whereby NKp46+NK cells express markers of memory-like/enhanced activation (increased expression of interferon gamma and Ly6C). Our data promotes a novel functional role for NKp46+NK cells in immunoprotection against experimental primary and secondary filarial infection which can proceed in the absence of adaptive immune regulation.

Lymphatic filariasis and onchocerciasis are major neglected tropical diseases affecting over 90 million people worldwide with painful and profoundly disfiguring pathologies (such as lymphoedema or blindness). Type 2 inflammation is a hallmark of filarial nematode tissue infection and is implicated both in eosinophil dependent immunity and lymphatic or ocular immunopathologies. Type-2 innate lymphoid cells (ILC2) are known to play an important role in the initiation of type 2 inflammation in helminth infection. We therefore tracked comparative IL-12Rβ2

Despite long-term mass drug administration programmes, approximately 220 million people are still infected with filariae in endemic regions. Several research studies have characterized host immune responses but a major obstacle for research on human filariae has been the inability to obtain adult worms which in turn has hindered analysis on infection kinetics and immune signalling. Although theLitomosoides sigmodontisfilarial mouse model is well-established, the complex immunological mechanisms associated with filarial control and disease progression remain unclear and translation to human infections is difficult, especially since human filarial infections in rodents are limited. To overcome these obstacles, we performed adoptive immune cell transfer experiments into RAG2IL-2Rγ-deficient C57BL/6 mice. These mice lack T, B and natural killer cells and are susceptible to infection with the human filariaLoa loa. In this study, we revealed a long-term release ofL. sigmodontisoffspring (microfilariae) in RAG2IL-2Rγ-deficient C57BL/6 mice, which contrasts to C57BL/6 mice which normally eliminate the parasites before patency. We further showed that CD4+T cells isolated from acuteL. sigmodontis-infected C57BL/6 donor mice or mice that already cleared the infection were able to eliminate the parasite and prevent inflammation at the site of infection. In addition, the clearance of the parasites was associated with Th17 polarization of the CD4+T cells. Consequently, adoptive transfer of immune cell subsets into RAG2IL-2Rγ-deficient C57BL/6 mice will provide an optimal platform to decipher characteristics of distinct immune cells that are crucial for the immunity against rodent and human filarial infections and moreover, might be useful for preclinical research, especially about the efficacy of macrofilaricidal drugs.

Background Current treatment options for onchocerciasis are sub-optimal, prompting research and development of a safe cure (macrofilaricide). Onchocerca ochengi, a parasite of cattle, is used as a close surrogate for the human parasite O. volvulus in a murine model for pre-clinical screening of macrofilaricides. Skin from naturally infected cattle have been used in previous studies as a reliable source of parasite material. However, there is limited knowledge on how source-related factors such as the microfilaridermia status of the cattle, the nodule load and nodular worm viability may affect survival of male O. ochengi worms implanted in the rodent hosts. Such relationships were investigated in this study. Methods Dermal tissue and nodules were obtained from Gudali cattle, dissected and cultured to obtain migrating microfilariae (mf) and male worms. Emerged male worms were implanted into SCID mice and Gerbils (Meriones unguiculatus) and recovery rates were determined upon 42 days post implantation. Finally, nodules were processed for histology and embryogram analyses to assess the nodular worm viability and fertility, respectively. Results Of the 69 cattle sampled, 24 (34.8%) were mf+ and 45 (65.2%) were mf–. The mean nodule loads were 180.5 ± 117.7 (mf+) and 110.6 ± 102.7 (mf-) (p = 0.0186). The mean male worm harvest from nodules were 76.8 ± 120.3 and 47.2 ± 33.4 (p = 0.2488) for mf+ and mf– cattle, respectively. The number of male worms per 100 nodules were 57/100 and 46/100 nodules for mf+ and mf– cows, respectively. Female worms from nodules of mf– cows had higher counts of both normal and abnormal embryos with higher proportions of dead nodular worms evinced by histology compared to those from mf+ cows. A total of 651 worms were implanted into mice and gerbils, out of which 129 (19.81%) were recovered. Logistic regression analysis indicated that the microfilaridermia status of the cattle (presence of mf) (OR = 4.3319; P = 0.001) is the single most important predictor of the success of male worm recovery after implantation into rodents. Conclusion Microfilaridermic cattle provide a promising source of adult O. ochengi. Male worms from this group of cattle have a better success rate of survival in a murine implant model. Nevertheless, in the programmatic point of view, amicrofilaridermic Gudali cattle would still constitute an important source of O. ochengi male worms with relatively good viability after implantation into rodents., Graphical abstract Image 1, Highlights • There is a need of developing safe macrofilaricide for human onchocerciasis using cattle Onchocerca ochengi. • Skin from naturally infected cattle have been used as a reliable parasite source for development of murine pre-clinical screening models. • The role of source-related factors on O. ochengi production and the success of pre-clinical screening models is assessed. • Worms from positive microfilaridermic cattle are successfully recovered after their implantation into rodents.

Background Infections with Onchocerca volvulus nematodes remain a threat in Sub-Saharan Africa after three decades of ivermectin mass drug administration. Despite this effort, there is still an urgent need for understanding the parasite biology especially the mating behaviour and nodule formation as well as the development of more potent drugs that can clear the developmental (L3, L4, L5) and adult stages of the parasite and inhibit parasite reproduction and behaviour. Methodology/Principal findings Prior to culture, freshly harvested O. volvulus L3 larvae from dissected Simulium damnosum flies were purified by centrifugation using a 30% Percoll solution to eliminate fly tissue debris and contaminants. Parasites were cultured in both cell-free and cell-based co-culture systems and monitored daily by microscopic visual inspection. Exhausted culture medium was replenished every 2–3 days. The cell-free culture system (DMEM supplemented with 10% NCS) supported the viability and motility of O. volvulus larvae for up to 84 days, while the co-culture system (DMEM supplemented with 10% FBS and seeded on LLC-MK2 feeder cells) extended worm survival for up to 315 days. Co-culture systems alone promoted two consecutive parasite moults (L3 to L4 and L4 to L5) with highest moulting rates (69.2±30%) observed in DMEM supplemented with 10% FBS and seeded on LLC-MK2 feeder cells, while no moult was observed in DMEM supplemented with 10% NCS and seeded on LEC feeder cells. In DMEM supplemented with 10% FBS and seeded on LLC-MK2 feeder cells, O. volvulus adult male worms attached to the vulva region of adult female worms and may have mated in vitro. Apparent early initiation of nodulogenesis was observed in both DMEM supplemented with 10% FBS and seeded on LLC-MK2 and DMEM supplemented with 10% NCS and seeded on LLC-MK2 systems. Conclusions/Significance The present study describes an in vitro system in which O. volvulus L3 larvae can be maintained in culture leading to the development of adult stages. Thus, this in vitro system may provide a platform to investigate mating behaviour and early stage of nodulogenesis of O. volvulus adult worms that can be used as additional targets for macrofilaricidal drug screening., Author summary River blindness affects people living in mostly remote and underserved rural communities in some of the poorest areas of the world. Although significant efforts have been achieved towards the reduction of disease morbidity, onchocerciasis still affects millions of people in Sub-Saharan Africa. The current control strategy is the annual mass administration of ivermectin which has accumulated several drawbacks over time, especially the action of the drug is solely microfilaricidal, very long treatment period (15–17 years) and reports of ivermectin losing its efficacy; thus, raising the urgent need for new adulticidal compounds. Our study has established an in vitro platform capable of supporting the growth and development of Onchocerca volvulus for up to 315 days, enabling the observation of parasite developmental processes: moulting (from the infective L3 stage to adults), increase in morphometry, the attachment of adult male and female worms and the potential initiation of nodulogenesis. Moreover, the platform might provide more insight into O. volvulus adult worms behavioural pattern in vitro. Also, our findings provide more avenues for mass production of different parasite stages, the investigation of parasite developmental biology and the identification of targets for drug discovery against different developmental stages of this filarial parasite within 315 days.

Basic and translational research on loiasis, a filarial nematode infection of medical importance, is impeded by a lack of suitable Loa loa infection models and techniques of obtaining and culturing life cycle stages. We describe the development of a new method for routine production of infective third-stage larvae (L3) of L. loa from the natural intermediate arthropod vector host, Chrysops silacea, following experimental infection with purified microfilariae. At 14-days post-infection of C. silacea, the fly survival rate was 43%. Survival was significantly higher in flies injected with 50 mf (55.2%) than those that received 100 mf (31.0%). However, yield per surviving fly and total yield of L3 was markedly higher in the group of flies inoculated with 100 mf (3474 vs 2462 L3 produced). The abdominal segment hosted the highest percentage recovery of L3 (47.7%) followed by head (34.5%) and thorax (17.9%). L. loa larval survival was higher than 90% after 30 days of in vitro culture. The in vitro moulting success rate to the L4 larval stage was 59.1%. After experimental infection of RAG2-/-IL-2γc-/—mice, the average L. loa juvenile adult worm recovery rate was 10.5% at 62 dpi. More than 87% of the worms were recovered from the muscles and subcutaneous tissues. Worms recovered measured an average 24.3 mm and 11.4 mm in length for females (n = 5) and males (n = 5), respectively. In conclusion, L. loa mf injected into C. silacea intrathoracically develop into infective larvae that remain viable and infective comparable to L3 obtained through natural feeding on the human host. This technique further advances the development of a full laboratory life cycle of L. loa where mf derived from experimentally-infected animals may be utilized to passage life cycle generations via intrathoracic injections of wild-caught vector hosts., Author summary The Neglected Tropical Disease, loiasis (tropical eye worm disease) affects 13–15 million individuals in Central Africa. Loiasis has recently been identified as a cause of premature mortality and is a barrier to ivermectin-based elimination of onchocerciasis or lymphatic filariasis where co-infections occur, due to the risk of serious adverse events. Loiasis lacks laboratory preclinical models for drug development, biomarker discovery and inflammatory pathology research. Here we detail the successful development of an experimental technique for the laboratory production of Loa loa infective larvae via injection of purified microfilariae into the thorax of wild-caught tabanid flies that are the natural transmission vector. The high yielding infective larvae produced in the laboratory were validated as biologically viable in culture and in a new mouse infection model whereby adult-stage parasites could be produced. The evidence reported herein is an important step to establishing a full laboratory life cycle of L. loa by passage between animal models and experimental injections of the wild-caught vector, Chrysops.

BackgroundInfections with Onchocerca volvulus nematodes remain a threat in Sub-Saharan Africa after two decades of ivermectin mass drug administration. Despite this effort, there is still an urgent need for understanding the parasite biology, especially mating behaviour and nodule formation, as well as development of more potent drugs that can clear the developmental (L3, L4, L5) and adult stages of the parasite and inhibit parasite’s reproductive and behavioural pattern.Methodology/Principal FindingsPrior to culture, freshly harvested O. volvulus L3 larvae from dissected Simulium were purified by centrifugation using a 30% Percoll solution to eliminate fly tissue debris and contaminants. Parasites were cultured in both cell-free and cell-based co-culture systems, and monitored daily by microscopic visual inspection. Exhausted culture medium was replenished every 2–3 days. The cell-free culture system supported the viability and motility of O. volvulus larvae for up to 84 days (DMEM–10%NCS), while the co-culture system (DMEM–10%FBS–LLC-MK2) extended the worm survival period to 315 days. Co-culture systems alone promoted the two consecutive parasite moults (L3 to L4 and L4 to L5) with highest moulting rates observed in DMEM–10%FBS–LLC-MK2 (69.2±30 %), while no moult was observed in DMEM–10%NCS–LEC condition. O. volvulus adult worms mating and even mating competitions were observed in DMEM–10% FBS –LLC-MK2 co-culture system. Early nodulogenesis was observed in both DMEM–10% FBS–LLC-MK2 and DMEM– 10%NCS–LLC-MK2 systems.Conclusions/SignificanceThe present study describes an in vitro system in which O. volvulus L3 larvae can be maintained in culture leading to the development of reproductive adult stages. Thus, this platform gives potential for the investigation of mating, mating competition and early stage of nodulogenesis of O. volvulus adult worms that can be used as additional targets for onchocercacidal drug screening.Author summaryRiver blindness affects people living in mostly remote and underserved rural communities in some of the poorest areas of the world. Although significant efforts have been achieved towards the reduction of disease morbidity, onchocerciasis still affect million of people in Sub-Saharan Africa. The current control strategy is the annual mass administration of ivermectin which have accumulated several drawbacks overtime: as the sole microfilaricidal action of the drug, very long treatment period (15-17 years) and reports of ivermectin losing its efficacy; Therefore, raising the urgent need for new onchocercacidal molecules. Our study has established an in vitro platform capable of supporting the growth and development of all developmental stages of O. volvulus (L3 infective stage, L4, L5 and adult worms), moreover the platform provided more insight on O. volvulus adult worms reproductive and behavioural pattern. Our findings provide more avenues for mass production of different parasite stages, the investigation of parasite developmental biology and the identification of targets for drug discovery against different phases of development of this filaria parasite

Background Different immune mechanisms are capable of killing developmental stages of filarial nematodes and these mechanisms are also likely to vary between the primary and a challenge infection. However, the lack of a detailed analysis of cytokine, chemokine and immunoglobulin levels in human loiasis is still evident. Therefore, detailed analysis of immune responses induced by the different developmental stages of Loa loa in immune-competent BALB/c mice will aid in the characterization of distinct immune responses that are important for the immunity against loiasis. Methods Different developmental stages of L. loa were obtained from human peripheral blood (microfilariae, MF), the transmitting vector, Chrysops (larval stage 3, L3) and infected immune-deficient BALB/cRAG2γc−/− mice (L4, L5, adult worms). Groups of wildtype BALB/c mice were then injected with the isolated stages and after 42 days post-infection (pi), systemic cytokine, chemokine and immunoglobulin levels were determined. These were then compared to L. loa-specific responses from in vitro re-stimulated splenocytes from individual mice. All parameters were determined using Luminex technology. Results In a pilot study, BALB/c mice cleared the different life stages of L. loa within 42 days pi and systemic cytokine, chemokine and immunoglobulin levels were equal between infected and naive mice. Nevertheless, L. loa-specific re-stimulation of splenocytes from mice infected with L5, MF or adult worms led to induction of Th2, Th17 and chemokine secretion patterns. Conclusions This study shows that although host immunity remains comparable to naive mice, clearance of L. loa life-cycle development stages can induce immune cell memory leading to cytokine, chemokine and immunoglobulins secretion patterns which might contribute to immunity and protection against reinfection.

Parasitic filarial nematodes cause debilitating infections in people in resource-limited countries. A clinically validated approach to eliminating worms uses a 4- to 6-week course of doxycycline that targetsWolbachia, a bacterial endosymbiont required for worm viability and reproduction. However, the prolonged length of therapy and contraindication in children and pregnant women have slowed adoption of this treatment. Here, we describe discovery and optimization of quinazolines CBR417 and CBR490 that, with a single dose, achieve >99% elimination ofWolbachiain the in vivoLitomosoides sigmodontisfilarial infection model. The efficacious quinazoline series was identified by pairing a primary cell-based high-content imaging screen with an orthogonal ex vivo validation assay to rapidly quantifyWolbachiaelimination inBrugia pahangifilarial ovaries. We screened 300,368 small molecules in the primary assay and identified 288 potent and selective hits. Of 134 primary hits tested, only 23.9% were active in the worm-based validation assay, 8 of which contained a quinazoline heterocycle core. Medicinal chemistry optimization generated quinazolines with excellent pharmacokinetic profiles in mice. Potent antiwolbachial activity was confirmed inL. sigmodontis,Brugia malayi, andOnchocerca ochengiin vivo preclinical models of filarial disease and in vitro selectivity againstLoa loa(a safety concern in endemic areas). The favorable efficacy and in vitro safety profiles of CBR490 and CBR417 further support these as clinical candidates for treatment of filarial infections.

Background Loiasis, an often-neglected tropical disease, is a threat to the success of lymphatic filariasis and onchocerciasis elimination programmes in rainforest areas of the central and western Africa. Its control and even its elimination might be possible through the use of a safe macrofilaricide, a prophylactic drug, or perhaps a vaccine. This present study evaluated the effect of flubendazole (FLBZ) on the development of Loa loa L3 in vitro and in vivo. Methods Infective stages of L. loa were isolated and co-cultured in Dulbecco’s Modified Eagle’s Medium in the presence of monkey kidney epithelial cells (LLC-MK2) feeder cells. FLBZ and its principal metabolites, reduced flubendazole (RFLBZ) and hydrolyzed flubendazole (HFLBZ), were screened in vitro at concentrations 0.05, 0.1, 0.5, 1 and 10 μg/ml. The viability of the parasites was assessed microscopically daily for 15 days. For in vivo study, a total of 48 CcR3 KO mice were infected subcutaneously with 200 L. loa L3 and treated with 10 mg/kg FLBZ once daily for 5 consecutive days. Twenty-four animals were used as control and received L3 and vehicle. They were dissected at 5, 10, 15 and 20 days post-treatment for worm recovery. Results The motility of L3 larvae in vitro was reduced from the second day of incubation with drugs at in vivo plasma concentration levels, with a strong correlation found between reduced motility and increased drug concentration (Spearman’s rho = -0.9, P < 0.0001). Except for HFLBZ (0.05 μg/ml and 0.01 μg/ml), all concentrations of FLBZ, HFLBZ and RFLBZ interrupted the moulting of L. loa infective larvae to L4. In vivo, regardless of the experimental group, there was a decrease in parasite recovery with time. However, at each time point this reduction was more pronounced in the group of animals treated with FLBZ compared to equivalent control. Parasites were recovered from the flubendazole-treated groups only on day 5 post-inoculation at an average rate of 2.1%, a value significantly lower (Mann-Whitney U-test, U = 28, P = 0.0156) than the average of 31.1% recovered from the control group. Conclusions This study reveals the ability of flubendazole to inhibit the development of L. loa L3 both in vitro and in vivo, and in addition validates the importance of in vitro and animal models of L. loa as tools for the development of drugs against loiasis. Electronic supplementary material The online version of this article (10.1186/s13071-018-3282-x) contains supplementary material, which is available to authorized users.

Elimination of the helminth disease, river blindness, remains challenging due to ivermectin treatment-associated adverse reactions in loiasis co-infected patients. Here, we address a deficit in preclinical research tools for filarial translational research by developing Loa loa mouse infection models. We demonstrate that adult Loa loa worms in subcutaneous tissues, circulating microfilariae (mf) and presence of filarial biomarkers in sera occur following experimental infections of lymphopenic mice deficient in interleukin (IL)-2/7 gamma-chain signaling. A microfilaraemic infection model is also achievable, utilizing immune-competent or -deficient mice infused with purified Loa mf. Ivermectin but not benzimidazole treatments induce rapid decline (>90%) in parasitaemias in microfilaraemic mice. We identify up-regulation of inflammatory markers associated with allergic type-2 immune responses and eosinophilia post-ivermectin treatment. Thus, we provide validation of murine research models to identify loiasis biomarkers, to counter-screen candidate river blindness cures and to interrogate the inflammatory etiology of loiasis ivermectin-associated adverse reactions., Here, the authors develop a mouse model of Loa loa that reflects human infections, including eosinophilia, and determine effects of ivermectin treatment.

The Onchocerca ochengi adult implant and Brugia malayi microfilariemic Severe-Combined Immunodeficient (SCID) mouse models are validated screens to measure macrofilaricidal and microfilaricidal activities of candidate onchocerciasis drugs. The purpose of this study was to assess whether 5 daily sub-cutaneous (s.c.) injections of standard flubendazole (FBZ) suspension (10mg/kg), a single s.c. injection (10mg/kg) or 5 daily repeated oral doses of FBZ amorphous solid dispersion (ASD) formulation (0.2, 1.5 or 15mg/kg) mediated macrofilaricidal efficacy against O. ochengi male worms implanted into SCID mice. The direct microfilaricidal activity against circulating B. malayi microfilariae of single dose FBZ ASD formulation (2 or 40 mg/kg) was also evaluated and compared against the standard microfilaricide, ivermectin (IVM). Systemic exposures of FBZ/FBZ metabolites achieved following dosing were measured by pharmacokinetic (PK) bioanalysis. At necropsy, five weeks following start of FBZ SC injections, there were significant reductions in burdens of motile O. ochengi worms following multiple injections (93%) or single injection (82%). Further, significant proportions of mice dosed following multiple injections (5/6; 83%) or single injection (6/10; 60%) were infection negative (drug-cured). In comparison, no significant reduction in recovery of motile adult O. ochengi adult worms was obtained in any multiple-oral dosage group. Single oral-dosed FBZ did not mediate any significant microfilaricidal activity against circulating B. malayi mf at 2 or 7 days compared with >80% efficacy of single dose IVM. In conclusion, multiple oral FBZ formulation doses, whilst achieving substantial bioavailability, do not emulate the efficacy delivered by the parenteral route in vivo against adult O. ochengi. PK analysis determined FBZ efficacy was related to sustained systemic drug levels rather than achievable Cmax. PK modelling predicted that oral FBZ would have to be given at low dose for up to 5 weeks in the mouse model to achieve a matching efficacious exposure profile., Author summary Onchocerciasis, caused by the filarial parasitic worm, Onchocerca volvulus, is a major cause of infection-related blindness and skin disease and is targeted for elimination. Current drugs are not optimal in all patient populations and a short-course cure would accelerate elimination. Flubendazole is used to treat intestinal worms in humans. The current oral formulation is not well absorbed into the blood. Flubendazole treatment has mediated curative efficacy in onchocerciasis patients when given as an injection but also causes adverse reactions at the injection site. In this study, we tested whether a new oral formulation of flubendazole with improved absorption could selectively kill Onchocerca adult parasites compared with circulating larval filarial parasites, using immunodeficient mouse models. Whilst injections of flubendazole mediated high levels of efficacy, five-day oral treatment, at a range of doses, did not emulate this curative effect. Oral flubendazole also did not significantly affect levels of microfilariae in the blood. After comparing levels of flubendazole in the blood following injection versus oral treatment, we conclude that a long duration, low dose of the oral formulation, for 5 weeks, would be needed to match the exposure of the drug following injection.